The biological rationale for the expression of two co-stimulatory molecules (CD80 and CD86) on the membrane of professional APC is not clear. Many results demonstrate that both molecules provide equivalent co-stimulatory signals to T-cells. Different roles for CD80 and CD86 in the activation of distinct subpopulations of T-cells have also been described in both in vivo and in vitro models. Our published data along with other results indicate that CD86, but not CD80, stimulates cellular immune responses during DNA vaccination of mice. The important question is why CD86, but not CD80 is required for augmentation of T-cell response after vaccination? Based on our results we suggest several mechanisms to explain these cell responses after vaccination? Based on our results we suggest several mechanisms to explain these functional differences between CD80 and CD86 molecules. First, expression of MHC class I and CD86, but not CD80 along with immunogen may induce local invasion of immunocompetent cells crucial for generation of immune responses. Second, CD80 and CD86 may activate different subpopulations of T-cells. Third, the triggering of receptors with CD80 and CD86 molecules may supply different signals to T-cells. Finally, the ligation of CD86, but not CD80 on the surface of APC could provide important signal to these cells and in turn induce production of certain molecules such as cytokines/lymphokines/chemokines, which are important for T cell activation. To evaluate these hypotheses we propose using DNA immunization as a model system to investigate the following: 1.) The differential effects of CD80 and CD86 expression in the microenvironment of MHC class I positive or negative muscle tissues (i.e. the invasion of immunocompetent cells at the site of DNA inoculation). 2). The activation of different subsets of CTL (CD4+ or C8+) and T helper cells (Th1 or Th2) after co-immunization with immunogen and CD80/CD86 expressing plasmids. 3). The functional region/s of human CD86 molecule involved in enhancement of cellular immune responses after co-immunization with DNA encoding immunogen and different CD80/CD86 mutant molecules. 4) The synergistic effect of co- stimulatory molecules and cytokines produced by professional APC, on T-cell activation after DNA vaccination. 5). The differential expression of genes after ligation of CD86 versus CD80 on muscle cells and professional APC.